bPTEN/SHIP^−/− B cells display a surface phenotype consistent with B cell lymphoma. (A) Percentage of CD19⁺ leukocytes in peripheral blood (PBL) and spleen of indicated mice, as determined by flow cytometry. pre = prelymphoma; bPS = bPTEN/SHIP^−/−. P-value was determined by two-tailed Student’s t test. Error bars represent SD of percentages of CD19⁺ B cells in blood or spleens of mice shown. (B) Flow cytometric analysis of PBL or spleen comparing expression of CD19 versus CD5. Numbers above gates indicate the percentage of CD19⁺ B cells in PBL or spleen, as indicated. (C) Flow cytometric analysis of CD19⁺ cells in PBL or single cell suspensions of spleen (top) using antibodies against cell surface markers, as indicated. The bottom shows a comparison of liver metastases and spleen of a diseased bPTEN/SHIP^−/− mouse. All histograms show gated CD19⁺ cells. All flow cytometry data are representative of five independent age-matched cohorts. WT, n = 10; bPTEN^−/−, n = 4; bSHIP^−/−, n = 9; bPTEN/SHIP^−/− pre, n = 5; bPTEN/SHIP^−/−, n = 6.

bPTEN/SHIP^−/− B cells induce disease upon transfer into TCR-βδ^−/− recipients. (A) bPTEN/SHIP^−/− or WT splenic B cells from age-matched mice were transferred intravenously into TCR-βδ^−/− recipients and donor cell accumulation in recipient peripheral blood assessed by flow cytometry at the indicated weeks. Shown are CD19⁺-gated PBL from four representative animals. Two received bPTEN/SHIP^−/− B cells and two received WT B cells. The bPTEN/SHIP^−/− mouse shown in the top panel died before 12 wk. (n = 8 WT recipients and 9 bPTEN/SHIP^−/− recipients). (B) The percentage of CD5⁺CD19⁺B220^low B cells in the peripheral blood of TCR-βδ^−/− animals after adoptive transfer of WT or bPTEN/SHIP^−/− B cells is shown over time. Each point represents a single animal. Error bars represent SD of percentages of CD5⁺CD19⁺B220^low PBLs in chimeric mice at each time point shown. (C) Flow cytometry plots of PBLs from representative chimeras stained for CD19, B220, CD11b, CD5, and IgD. Data shown are 9 wk after transfer. (D) Kaplan-Meier survival curve of TCR-βδ^−/− recipients after adoptive transfer of WT or bPTEN/SHIP^−/− B cells. WT, n = 8; bPTEN/SHIP^−/−, n = 9.

bPTEN/SHIP^−/− B cells survive better than WT, bPTEN^−/−, or bSHIP^−/− B cells in vitro. (A) WT, bPTEN^−/−, bSHIP^−/−, or bPTEN/SHIP^−/− B cells were cultured at a starting concentration of 5 × 10⁵ cells/ml in media lacking any growth factors and surviving cells were counted every 24 h using a Coulter counter. Data are representative of n = 3 independent experiments. Error bars represent SD of total live cells of triplicate wells counted in triplicate for each mouse. (B) As in A, with the modification that cell viability was assessed by annexin V (Ann V) and propidium iodide (PI) staining. (Top) Shown is a representative panel of WT, bPTEN^−/−, bSHIP^−/−, and bPTEN/SHIP^−/− at 72 h (top) and a graph showing the percentage of live (Ann V-PI⁻) cells over time (bottom). Data are representative of n = 3 independent experiments. Error bars represent SD of the percentage of annexin V–propidium iodide (Ann V-PI⁻) B cells for WT, bPTEN^−/−, bSHIP^−/−, and bPTEN/SHIP^−/− mice at each time point shown. (C) As in A, with the modification that cells were lysed at indicated time points and Western blotting performed to detect levels of pAkt (Ser 473), total Akt, pGSK3β (Ser 9), and MCL-1. Total ERK1/2 levels were used as a loading control. Data are representative of n = 3 independent experiments. (D) Bim expression in freshly isolated splenic WT, bPTEN^−/−, bSHIP^−/−, or bPTEN/SHIP^−/− B cells was determined by immunoblotting with anti-Bim. Blots were stripped and reprobed with anti-actin antibodies as a loading control. Data are representative of n = 3 experiments.

Human DLBCL patients exhibit reduced PTEN and SHIP expression compared with FL patients. (A) cDNA expression analysis of human malignant B cell lymphoma samples from publicly available patient data (www.oncomine.org) comparing normal B cells, FL, and DLBCL showing mean (black lines) relative expression values for PTEN and SHIP cDNA. Each square represents an expression value for a single patient/sample. P-values comparing normal cells and DLBCL cells are indicated. (B) Mean Log₂ normalized expression of PTEN and SHIP is plotted for the following patient groups: PTEN low (lowest 25% PTEN expression), SHIP low (lowest 25% SHIP expression), and BCL6 high (highest 25% BCL6 expression), in comparison with mean values for all patients which were near 0. Analyses of other genes in the PTEN and SHIP low patient cohort confirmed that results were not a result of generally low cDNA signals in this cohort. *, P < 0.05; **, P < 0.005.

bPTEN/SHIP^−/− mice develop a lethal disease characterized by weight loss, splenomegaly, and altered splenic architecture. (A) Spleen (left axis) and body (right axis) weight (grams) of WT (black bar) and bPTEN/SHIP^−/− (white bar) mice between 7 and 12 mo of age (n = 7). Error bars represent SD of spleen and body weights of WT or bPTEN/SHIP^−/− mice shown. (B) Representative photograph of spleens from WT (12 mo), bPTEN^−/− (12 mo), bSHIP^−/− (12 mo), and bPTEN/SHIP^−/− (8 mo) mice. Image is representative of n > 25 mice of each genotype. (C) Survival of WT (n = 20), bPTEN^−/− (n = 15), bSHIP^−/− (n = 15), and bPTEN/SHIP^−/− (n = 20) mice monitored for 1 yr. (D) Hemotoxylin/eosin (H&E) staining of microsections from paraffin-embedded spleens. The age of mice shown was 4 or 10 mo, as indicated. Pictures are representative of >40 mice of each genotype. Magnification shown is with a 5× objective. Bars, 500 µm. (E) Immunofluorescence of spleens of WT and bPTEN/SHIP^−/− mice of indicated ages stained with α-CD19-PE (red), α-CD11b-FITC (green), and F4/80-APC (blue). Magnification shown is 5×. Bars, 500 µm. (F) H&E-stained sections from paraffin-embedded liver (top) and lung (bottom) of WT and bPTEN/SHIP^−/− mice. Arrows indicate white blood cell infiltrates. Results are representative of >20 WT and bPTEN/SHIP^−/− mice >6 mo of age. Magnification shown is with a 5× objective (first and third rows) or 40× objective (second and fourth rows). Scale bars: (5×) 500 µm; (40×) 100 µm.

bPTEN/SHIP^−/− B cells have oligoclonal Ig repertoires. (A) Rearrangements of the heavy chain of WT and bPTEN/SHIP^−/− B cells were detected using the pJ11 probe, which preferentially detects rearrangements to J_H1, J_H2, and J_H3. All mice were >6 mo of age and B cells were isolated from spleen. Pre-lymph. = prelymphoma. Clonal rearrangements are indicated by ellipses. Each lane represents an individual mouse. (B) PCR analysis of Ig V-DJ_H recombination using degenerate 5′ primers specific for either the distal J558 (V_H1) or the proximal 7183 (V_H5) family and a common 3′ J_H4 primer on DNA from spleen (sp), liver (li), and/or lung (lu) from WT or bPTEN/SHIP^−/− mice. Arrows indicate amplified V-DJ_H1, V-DJ_H2, and V-DJ_H3 segment recombinations (1.3, 0.95, and 0.4 kb, respectively). Representative samples are shown for WT and young (<6 mo) and old (>6 mo) bPTEN/SHIP^−/− mice.

bPTEN/SHIP^−/− B cells proliferate in response to treatment with BAFF. (A) Purified splenic B cells from WT, bPTEN^−/−, bSHIP^−/−, or bPTEN/SHIP^−/− mice were stimulated with anti-IgM F(ab’)₂ (α-IgM) in the presence or absence of BAFF or with APRIL as indicated. Proliferation was determined at 48 h by ³H-thymidine incorporation. Y-axis values shown are ×10⁻³ cpm. All assays were conducted in triplicates and SDs are shown as error bars. The results are representative of six experiments. (B) Western blots of protein lysates from WT, bPTEN^−/− (P), bSHIP^−/− (S), or bPTEN/SHIP^−/− (PS) splenic B cells either freshly isolated (t = 0) or stimulated for 48 h, as indicated, were probed with anti-p27^kip1, anti-cyclin D3, or anti-actin antibodies. (C) WT, bPTEN^−/−, bSHIP^−/−, or bPTEN/SHIP^−/− B cells were stimulated with anti-IgM F(ab’)₂ (α-IgM), BAFF, or anti-IgM F(ab’)₂ (α-IgM) + BAFF for the indicated time points. Western blots were probed with antibodies recognizing the phosphorylated form of Akt (Ser 473). Total ERK1/2 was used as a loading control. For B and C, data are representative of n = 5 independent experiments.

bPTEN/SHIP^−/− mice develop different B cell neoplasias. (A) Representative examples of B-NHL developed by bPTEN/SHIP^−/− mice. (1) Normal spleen (10×): white pulp follicle filled with small lymphocytes surrounded by an MZ and red pulp. (2) MZL (10×): white pulp with a periarteriolar lymphoid sheath of small lymphocytes surrounded by an expanse of uniform medium-sized MZ B cells extending into the red pulp. (3) Centroblastic lymphoma (100×): white pulp is taken over by large blasts with dispersed chromatin and one or more nucleoli, often located near the nuclear membrane. One mitotic figure is seen near the center, and multiple apoptotic bodies are present. (4) Follicular lymphoma (100×): the white pulp is occupied by a heterogeneous population of smaller centrocytes with angulated and folded nuclei as well as larger centroblasts with more dispersed chromatin and peripheral nucleoli. (5) Plasmacytoma (100×): lymph node populated with plasmablasts with thick nuclear membranes and prominent nucleoli. Plasmacytic cells with relatively condensed chromatin and central nucleoli, and a prominent mitotic figure are also present. (6) Histiocyte-associated DLBCL (100×): histiocytes with very large nuclei with reticular chromatin and irregular nucleoli are present along with an abundance of foamy pink cytoplasm. One mitotic figure can be seen in the top right. The lymphoid population is comprised of centroblasts and several immunoblasts. Bars: (1 and 2) 500 µm; (3–6) 50 µm. (B) Classification of B-NHL displayed by bPTEN/SHIP^−/− mice as determined by histological examination of splenic samples. CBL, centroblastic lymphoma; PCT, plasmacytoma; FL, follicular lymphoma. The chart represents 15 different animals per group. (C) Relative levels of PTEN and SHIP mRNA transcripts in mouse B cell lymphoma determined by oligo-microarray. Values from 10–15 mice in each lymphoma group were tested against normal spleen by Mann-Whitney U test. The pink line shows the median in each group. CBL, centroblastic lymphoma; IBL, immunoblastic lymphoma; NOR, normal spleen. The mean Log₂ normalized expression of PTEN and SHIP is plotted. P-values are indicated.